1. Field of the Invention
This invention relates to antisense oligonucleotides which modulate the activity of the ribonucleotide reductase genes and the secA genes in microorganisms. This invention is also related to methods of using such compounds in inhibiting the growth of microorganisms.
These antisense oligonucleotides are particularly useful in treating pathological conditions in mammals which are mediated by the growth of microorganisms. Accordingly, this invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable excipient and an effective amount of a compound of this invention.
These antisense oligonucleotides may also be used as anti-microbial agents for agricultural applications such as crop protection.
2. References
The following publications, patent applications and patents are cited in this application as superscript numbers:
1. Nordlund and Eklund xe2x80x9cStructure and function of the Escherichia coli ribonucleotide reductase protein R2xe2x80x9d, J. Mol. Biol. (1993) 232:123-164;
2. Carlson et al., xe2x80x9cPrimary structure of the Escherichia coli ribonucleoside diphosphate reductase operonxe2x80x9d, PNAS USA (1984) 81:4294-4297;
3. Nilsson et al., xe2x80x9cNucleotide sequence of the gene coding for the large subunit of ribonucleotide reductase of Escherichia coli Correctionxe2x80x9d, Nucleic Acids Research (1988) 16:4174;
4. P. Reichard, xe2x80x9cThe anaerobic ribonucleotide reductase from Escherichia colixe2x80x9d, J. Biol. Chem. (1993) 268:8383-8386;
5. Nordlund et al., Nature (1990) 345:593-598;
6. der Blaauwen et al., xe2x80x9cInhibition of preprotein translocation and reversion of the membrane inserted state of secA by a carboxyl terminus binding Mabxe2x80x9d, Biochemistry (1997) 36:9159-9168;
7. McNicholas et al., xe2x80x9cDual regulation of Escherichia coli secA translation by distinct upstream elementsxe2x80x9d, J. Mol. Biol. (1997) 265:128-141;
8. U.S. Pat. No. 5,294,533;
9. Gasparro et al., xe2x80x9cPhotoactivatable antisense DNA: Suppression of ampicillin resistance in normally resistant Escherichia colixe2x80x9d, Antisense Research and Development (1991) 1:117-140;
10. White et al., xe2x80x9cInhibition of the multiple antibiotic resistance (mar) operon in Escherichia coli by antisense DNA analogsxe2x80x9d, Antimicrobial Agents and Chemotherapy (1997) 41:2699-2704;
11. Nielsen et al., Science (1991) 354:1497;
12. Good and Nielsen, xe2x80x9cInhibition of translation and bacterial growth by peptide nucleic acid targeted to ribosomal RNAxe2x80x9d, PNAS USA (1998) 95:2073-2076;
13. Buchardt, deceased, et al., U.S. Pat. No. 5,766,855;
14. Buchardt, deceased, et al., U.S. Pat. No. 5,719,262;
15. U.S. Pat. No. 5,034,506;
16. Altschul, et al., xe2x80x9cBasic local alignment search toolxe2x80x9d, J. Mol. Biol. (1990) 215:403-10;
17. Devereux. et al., xe2x80x9cA comprehensive set of sequence analysis programs for the VAXxe2x80x9d, Nucleic Acids Res. (1984) 12:387-395;
18. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989, 1992);
19. Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore Md. (1989);
20. Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor Mich. (1995);
21. Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995);
22. Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988)
23. U.S. Pat. No. 5,023,252, issued June 11, 1991
24. Felgner et al., U.S. Pat. No. 5,580,859.
25. U.S. Pat. No. 5,011,472
26. Remington""s Pharmaceutical Sciences, Mace Publishing Company, Philadelphia Pa. 17th ed. (1985);
27. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons, New York (1988).
28. PCR Protocols: A Guide To Methods And Applications, Academic Press, San Diego, Calif. (1990).
29. Dower, W. J., Nucleic Acids Res. (1988) 16:6127;
30. Neuman et al., EMBO J. (1982) 1:841;
31. Taketo A., Biochim Biophys. Acta (1988) 949:318;
32. Miller J. H. Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1972);
33. Horwitz J. P., J. Med. Chem. (1964) 7:574;
34. Mann et al., Biochem.(1991) 30:1939;
35. Olsvik, et al., Acta Pathol. Microbiol. Immunol. Scand. [B] (1982) 90:319;
36. Laemmli, U. K., Nature (1970) 227:680;
37. Choy et al., Cancer Res.(1988) 48:2029;
38. Wright and Anazodo, Cancer J. (1988) 8:185-189;
39. Chan et al., Biochemistry (1993) 32:12835-12840;
40. Carpentier P. L., Microbiology 4th ed. W.B.Saunders Company (1977); and
41. Wright et al., Adv. Enzyme Regul. (1981) 19:105-127.
All of the above publications, patent applications and patents are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.
3. State of the Art
Ribonucleotide reductase catalyzes the de novo production of deoxyribonucleotides. The enzyme reduces the four main ribonucleotides to the corresponding deoxyribonucleotides required for DNA synthesis and repair (Wright et al.41).
In mammalian and bacterial cells, de novo production of deoxyribonucleotides by ribonucleotide reductase is usually highly regulated on different levels in order to produce the correct amount of deoxyribonucleotides for DNA synthesis. In the DNA viruses, the metabolism of the host cell is directed towards production of viral DNA by virus encoded ribonucleotide reductases (Nordlund and Eklund1).
Mammalian cells and many DNA viruses and prokaryotes, have a heterodimeric iron-containing ribonucleotide reductase enzyme of the xcex12xcex22 type. For example, ribonucleotide reductase from E. coli is a multi-subunit xcex12xcex22 enzyme where the two homo-dimeric proteins are denoted R1 and R2. The larger xcex12 protein, R1, contains the binding sites for substrate and allosteric effectors and also the redox-active cysteine residues. Protein R1 has a molecular mass of 2xc3x9786,000 where each subunit contains 761 residues. The smaller xcex22 protein, denoted R2, contains the dinuclear ferric center and a stable free tyrosyl radical necessary for the enzymatic activity. The R2 protein has a molecular mass of 2xc3x9743,500, where each subunit contains 375 amino acid residues (Nordlund and Eklund1).
The nucleotide sequence of the E. coli K-12 DNA comprising the operon for the structural genes of the subunits of ribonucleotide reductase has been determined. The DNA sequence includes a total length of 8557 nucleotides. An open reading frame between nucleotides 3506 and 5834 has been identified as the nrdA gene. An open reading frame between nucleotides 6012 and 7139 encoding a 375-amino acid polypeptide has been identified as the nrdB gene (Carlson et al.2, and Nilsson et al.3). The sequences of the nrdA and nrdB genes for E. coli are shown in FIGS. 1 and 2.
In E. coli, the synthesis of ribonucleotide reductase is controlled at the level of transcription. The nrdA and nrdB genes direct the synthesis of a 3.2 kilobase polycistronic mRNA. Perturbations in DNA replication, either a shift up in growth conditions or an inhibition of DNA synthesis leads to increased synthesis of nrd MRNA (Carlson et al.2).
A separate anaerobic ribonucleotide reductase has also been identified from E.coli. The anaerobic E. coli reductase has a molecular mass of 145 kD and is a homodimer. The gene for the anaerobic reductase (nrdD) has been cloned and sequenced (P. Reichard4).
The ribonucleotide reductase R2 genomic or cDNA sequences are known for several other species such as bacteriophage T4, clam, mouse, Saccharomyces cerevisiae, vaccinia, herpes simplex virus types 1 and 2, varicella and Epstein-Barr virus (Nordlund et al.5). The sequence of the nrdE and nrdF which code for the ribonucleotide reductase genes of S. typhimurium are shown in FIG. 3. The sequence of the ribonucleotide reductase gene of Lactococcus lactis is shown in FIG. 4.
The secA gene of E. coli encodes for one component of a multi-component system for the secretion of proteins across the inner membrane of E. coli (der Blaauwen et al.6). The complete system consists of the SecB protein, a cytosolic chaperone, the SecA protein, the translocation ATPase and the heterotrimeric integral membrane SecY/SecE/SecG complex, which along with SecA serves as the preprotein channel. SecA protein plays a central role in the secretion process by binding the preprotein, secB protein, anionic phospholipids and SecY/SecE/SecG protein. These interactions allow SecA to recognize soluble preprotein and recruit it to translocation sites in the inner membrane. Once such protein translocation complexes have assembled; further steps require an ATP-driven cycle of insertion and de-insertion of secA protein with the inner membrane, where each cycle appears to be coupled to the translocation of a segment of the preprotein.
SecA is the only component of the secretion apparatus that has been shown to be regulated. SecA is the second gene in the geneX-secA operon and its translation varies over a tenfold range depending on the status of protein secretion in the cell. During protein-export proficient conditions secA auto-represses its translation by binding to a site that overlaps the secA ribosome-binding site of genes-secA RNA. SecA protein can also dissociate a preformed 30 S-tRNAMET-genes-secA RNA ternary complex in vitro. However, during a protein export block secA translation increases substantially although the mechanism responsible for this regulatory response has not been elucidated (McNicholas et al.7). The sequence of the secA gene of E. coli is shown in FIG. 5.
The secA gene sequence has been identified for a number of other species including Mycobacterium bovis (FIG. 6), Mycobacterium tuberculosis (FIG. 7), Staphylococcus aureus (FIG. 8), Staphylococcus carnosus (FIG. 9), Bacillus subtilis, Bacillus firnus, Listeria monocytogenes, Mycobacterium smegmatis, Borrelia burgdorferi, P. sativum, S. griseus, and Synechoccus sp.
Antibiotics are important pharmaceuticals for the treatment of infectious diseases in a variety of animals including man. The tremendous utility and efficacy of antibiotics results from the interruption of bacterial (prokaryotic) cell growth with minimal damage or side effects to the eukaryotic host harboring the pathogenic organisms. In general, antibiotics destroy bacteria by interfering with the DNA replication, DNA to RNA transcription, translation (that is RNA to protein) or cell wall synthesis.
Although bacterial antibiotic resistance has been recognized since the advent of antimicrobial agents, the consequence of the emergence of resistant microorganisms, such resistance was historically controlled by the continued availability of effective alternative drugs. Now, drug resistance has emerged as a serious medical problem in the community, leading to increasing morbidity and mortality. The problem is worsened by the growing number of pathogens resistant to multiple, structurally unrelated drugs. The situation has become so desperate that antibiotics once removed from use because of toxic effects may be prescribed in an attempt to deal with the otherwise untreatable drug resistant bacteria.
Antisense oligonucleotides have been used to decrease the expression of specific genes by inhibiting transcription or translation of the desired gene and thereby achieving a phenotypic effect based upon the expression of that gene (Wright and Anazado38). For example, antisense RNA is important in plasmid DNA copy number control, in development of bacteriophage P22. Antisense RNA""s have been used experimentally to specifically inhibit in vitro translation of mRNA coding specifically from Drosophila hsp23, to inhibit Rous sarcoma virus replication and to inhibit 3T3 cell proliferation when directed toward the oncogene c-fos. Furthermore, it is not necessary to use the entire antisense MRNA since a short antisense oligonucleotide can inhibit gene expression. This is seen in the inhibition of chloramphenicol acetyltransferase gene expression and in the inhibition of specific antiviral activity to vesicular stomatitus virus by inhibiting the N-protein initiation site. Antisense oligonucleotides directed to the macromolecular synthesis operon of bacteria, containing the rpsU gene, the rpoD gene and the dnaG gene have been used for the detection of bacteria. (U.S. Pat. No. 5,294,5338). Furthermore, photoactivatable antisense DNA complementary to a segment of the P-lactamase gene has been used to suppress ampicillin resistance in normally resistant E. coli (Gasparro et al.9). Antisense DNA analogs have also been used to inhibit the multiple antibiotic resistant (mar) operon in Escherichia coli (White et al.10).
Accordingly, there is a need to develop antisense oligonucleotides which will act to inhibit the growth of microorganisms.
This invention is directed to antisense oligonucleotides which modulate the expression of the ribonucleotide reductase and secA genes in microorganisms and pharmaceutical compositions comprising such antisense oligonucleotides. This invention is also related to methods of using such antisense oligonucleotides for inhibiting the growth of microorganisms.
Accordingly, in one of its composition aspects, this invention is directed to an antisense oligonucleotide, which oligonucleotide is nuclease resistant and comprises from about 3 to about 50 nucleotides, which nucleotides are complementary to the ribonucleotide reductase gene or the secA gene of a microorganism. The antisense oligonucleotide may have one or more phosphorothioate internucleotide linkages.
In another of its composition aspects, this invention is directed to an antisense oligonucleotide comprising from about 3 to about 50 nucleotides which is capable of binding to the ribonucleotide reductase gene or the secA gene of a microorganism, wherein the oligonucleotide comprises all or part of a sequence selected from the group consisting of SEQ ID NO:22; SEQ ID NO:43; SEQ ID NO:62; SEQ ID NO:74; SEQ ID NO:75; SEQ ID NO:76; SEQ ID NO:143; SEQ ID NO:145; SEQ ID NO:152; SEQ ID NO:164; SEQ ID NO:176; SEQ ID NO:186; SEQ ID NO:188; SEQ ID NO:189; SEQ ID NO: 191; SEQ ID NO: 192; SEQ ID NO:195; SEQ ID NO:197; SEQ ID NO:206; SEQ ID NO:212; SEQ ID NO:220; SEQ ID NO:229; SEQ ID NO:235; SEQ ID NO:254; SEQ ID NO:261; SEQ ID NO:262; SEQ ID NO:263; SEQ ID NO:264; and SEQ ID NO:265.
In still another of its composition aspects, this invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of an antisense oligonucleotide, which oligonucleotide is nuclease resistant and comprises from about 3 to about 50 nucleotides, which nucleotides are complementary to the ribonucleotide reductase gene or the secA gene of a microorganism. The oligonucleotide may be modified, for example, the oligonucleotide may have one or more phosphorothioate internucleotide linkages.
In one of its method aspects, this invention is directed to a method for inhibiting the expression of the ribonucleotide reductase gene in a microorganism having a ribonucleotide reductase gene comprising, administering to said microorganism or to a cell infected with said microorganism an effective amount of an antisense oligonucleotide comprising from at least about 3 nucleotides which are complementary to the ribonucleotide reductase gene of the microorganism under conditions such that the expression of the ribonucleotide reductase gene is inhibited.
In another of its method aspects, this invention is directed to a method for inhibiting the expression of the secA gene in a microorganism having a secA gene, comprising administering to said microorganism an effective amount of an antisense oligonucleotide comprising from at least about 3 nucleotides which are complementary to the secA gene of the microorganism under conditions such that expression of the secA gene is inhibited.
In one of its method aspects, this invention is directed to a method for inhibiting the growth of a microorganism encoding a ribonucleotide reductase gene or a secA gene, which method comprises administering to said microorganism or a cell infected with said microorganism an effective amount of an antisense oligonucleotide comprising from at least about 3 nucleotides which are complementary to either the ribonucleotide reductase gene or the secA gene of the microorganism under conditions such that the growth of the microorganism is inhibited. Preferably, the antisense oligonucleotide is selected from the group consisting of SEQ ID NO:22; SEQ ID NO:43; SEQ ID NO:62; SEQ ID NO:74; SEQ ID NO:75; SEQ ID NO:76; SEQ ID NO:143; SEQ ID NO:145; SEQ ID NO:152; SEQ ID NO:164; SEQ ID NO:176; SEQ ID NO:186; SEQ ID NO:188; SEQ ID NO:189; SEQ ID NO:191; SEQ ID NO:192; SEQ ID NO:195; SEQ ID NO:197; SEQ ID NO:206; SEQ ID NO:212; SEQ ID NO:220; SEQ ID NO:229; SEQ ID NO:235; SEQ ID NO:254; SEQ ID NO:261; SEQ ID NO:262; SEQ ID NO:263; SEQ ID NO:264; and SEQ ID NO:265.
In another of its method aspects, this invention is directed to a method for treating a mammalian pathologic condition mediated by a microorganism, which method comprises identifying a mammal having a pathologic condition mediated by a microorganism having a ribonucleotide reductase gene or a secA gene and administering to said mammal an effective amount of an antisense oligonucleotide comprising from at least about 3 nucleotides which are complementary to either the ribonucleotide reductase gene or the secA gene of the microorganism under conditions such that the growth of the microorganism is inhibited.